Transcriptional regulation is an essential aspect of cell growth and differentiation, as well as critical in the response of cells to environmental changes. In humans, defects in transcription often lead to disease. Therefore, the elucidation of transcriptional mechanisms is important for understanding human biology. This proposal focuses on two proteins, Spt6 and Iws1, that are conserved from yeast to humans, that are essential for viability, and that have been implicated in cancer. Spt6 and Iws1 function as global transcriptional regulators and control gene expression at multiple levels. Despite intensive studies, the exact mechanisms by which Spt6/Iws1 regulates transcription is poorly understood. To investigate the roles that Spt6 and Iws1 play in gene expression the following specific aims are proposed: Aim 1. Elucidate the in vivo roles of the conserved motifs of the Iws1 protein. Using site- directed mutagenesis of the conserved residues, isolation of conditionally lethal mutants, and a protein depletion approach, the functions of Iws1 will be analyzed. The proposed mutant analysis will reveal the functions of Iws1 in transcription, mRNA processing, protein-protein interactions, and recruitment of critical factors to chromatin genome-wide. Aim 2. Isolate and characterize suppressors of spn1? and the spt6? null alleles. A genetic screen for suppressors of iws1? and spt6? inviability will be performed to discover novel cellular activitie and pathways that can compensate for loss of Spt6 and Iws1. Detailed characterization of the identified suppressors, including several mutations already identified in the pilot screen, will shed light on the mechanisms by which these mutants rescue absence of Iws1 and will expand our understanding on the roles of Spt6 and Iws1 in gene expression. Altogether, the proposed studies will further our current understanding of the essential functions of Spt6 and Iws1 in transcriptional regulation. Our long-term goal is to understand the cascade of events and the contribution of each activity in gene expression control. Relevance: Deregulation of gene expression is tightly linked to tumorigenesis. Given the conserved nature of the studied factors and their roles in development and oncogenesis, our studies using yeast as model organisms will be broadly applicable to other eukaryotes. This research project has a long-term implication in anticancer drug discovery.